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- EMDB-8354: Cryo-EM structure of Polycystic Kidney Disease protein 2 (PKD2), ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8354
TitleCryo-EM structure of Polycystic Kidney Disease protein 2 (PKD2), residues 198-703
Map dataB-factor sharpened, masked map generated from relion auto-refinement and postprocessing
Sample
  • Complex: hPKD:198-703
    • Protein or peptide: hPKD:198-703, Polycystin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


detection of nodal flow / metanephric smooth muscle tissue development / metanephric cortex development / metanephric cortical collecting duct development / metanephric distal tubule development / polycystin complex / mesonephric tubule development / mesonephric duct development / : / metanephric part of ureteric bud development ...detection of nodal flow / metanephric smooth muscle tissue development / metanephric cortex development / metanephric cortical collecting duct development / metanephric distal tubule development / polycystin complex / mesonephric tubule development / mesonephric duct development / : / metanephric part of ureteric bud development / determination of liver left/right asymmetry / renal tubule morphogenesis / metanephric ascending thin limb development / HLH domain binding / basal cortex / metanephric mesenchyme development / metanephric S-shaped body morphogenesis / renal artery morphogenesis / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / cilium organization / VxPx cargo-targeting to cilium / detection of mechanical stimulus / regulation of calcium ion import / cation channel complex / calcium-induced calcium release activity / placenta blood vessel development / muscle alpha-actinin binding / voltage-gated monoatomic ion channel activity / cellular response to hydrostatic pressure / outward rectifier potassium channel activity / voltage-gated monoatomic cation channel activity / non-motile cilium / cellular response to fluid shear stress / cellular response to osmotic stress / voltage-gated sodium channel activity / inorganic cation transmembrane transport / transcription regulator inhibitor activity / actinin binding / motile cilium / determination of left/right symmetry / protein heterotetramerization / neural tube development / aorta development / ciliary membrane / branching involved in ureteric bud morphogenesis / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / heart looping / voltage-gated potassium channel activity / cytoplasmic side of endoplasmic reticulum membrane / cell surface receptor signaling pathway via JAK-STAT / potassium channel activity / centrosome duplication / sodium ion transmembrane transport / negative regulation of ryanodine-sensitive calcium-release channel activity / voltage-gated calcium channel activity / embryonic placenta development / monoatomic cation channel activity / release of sequestered calcium ion into cytosol / cellular response to cAMP / potassium ion transmembrane transport / cellular response to calcium ion / cytoskeletal protein binding / basal plasma membrane / ciliary basal body / liver development / establishment of localization in cell / lumenal side of endoplasmic reticulum membrane / calcium ion transmembrane transport / protein tetramerization / cytoplasmic vesicle membrane / cilium / intracellular calcium ion homeostasis / Wnt signaling pathway / cellular response to reactive oxygen species / phosphoprotein binding / mitotic spindle / calcium ion transport / positive regulation of nitric oxide biosynthetic process / cell-cell junction / lamellipodium / regulation of cell population proliferation / heart development / ATPase binding / positive regulation of cytosolic calcium ion concentration / protein homotetramerization / basolateral plasma membrane / transmembrane transporter binding / regulation of cell cycle / negative regulation of cell population proliferation / signaling receptor binding / calcium ion binding / endoplasmic reticulum membrane / positive regulation of gene expression / Golgi apparatus / endoplasmic reticulum / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular exosome / membrane
Similarity search - Function
Ferredoxin I 4Fe-4S cluster domain / Polycystin domain / Polycystin domain / Polycystic kidney disease type 2 protein / Polycystin cation channel, PKD1/PKD2 / Polycystin cation channel / Voltage-dependent channel domain superfamily / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsShen PS / Yang X / DeCaen PG / Liu X / Bulkley D / Clapham DE / Cao E
CitationJournal: Cell / Year: 2016
Title: The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs.
Authors: Peter S Shen / Xiaoyong Yang / Paul G DeCaen / Xiaowen Liu / David Bulkley / David E Clapham / Erhu Cao /
Abstract: The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM ...The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.
History
DepositionSep 27, 2016-
Header (metadata) releaseOct 26, 2016-
Map releaseNov 2, 2016-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5t4d
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8354.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor sharpened, masked map generated from relion auto-refinement and postprocessing
Voxel sizeX=Y=Z: 1.2156 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.21346551 - 0.40400645
Average (Standard dev.)0.0009170965 (±0.013227765)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 233.3952 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21559895833331.21559895833331.2155989583333
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z233.395233.395233.395
α/β/γ90.00090.00090.000
start NX/NY/NZ-54-3-120
NX/NY/NZ12323205
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2130.4040.001

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Supplemental data

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Mask #1

Fileemd_8354_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: RELION half map 2 (relion refine)

Fileemd_8354_half_map_1.map
AnnotationRELION half map 2 (relion_refine)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: RELION half map 1 (relion refine)

Fileemd_8354_half_map_2.map
AnnotationRELION half map 1 (relion_refine)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : hPKD:198-703

EntireName: hPKD:198-703
Components
  • Complex: hPKD:198-703
    • Protein or peptide: hPKD:198-703, Polycystin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: hPKD:198-703

SupramoleculeName: hPKD:198-703 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI-/- / Recombinant plasmid: pFastbac1

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Macromolecule #1: hPKD:198-703, Polycystin-2

MacromoleculeName: hPKD:198-703, Polycystin-2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 59.429145 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GAMGSRGLWG TRLMEESSTN REKYLKSVLR ELVTYLLFLI VLCILTYGMM SSNVYYYTRM MSQLFLDTPV SKTEKTNFKT LSSMEDFWK FTEGSLLDGL YWKMQPSNQT EADNRSFIFY ENLLLGVPRI RQLRVRNGSC SIPQDLRDEI KECYDVYSVS S EDRAPFGP ...String:
GAMGSRGLWG TRLMEESSTN REKYLKSVLR ELVTYLLFLI VLCILTYGMM SSNVYYYTRM MSQLFLDTPV SKTEKTNFKT LSSMEDFWK FTEGSLLDGL YWKMQPSNQT EADNRSFIFY ENLLLGVPRI RQLRVRNGSC SIPQDLRDEI KECYDVYSVS S EDRAPFGP RNGTAWIYTS EKDLNGSSHW GIIATYSGAG YYLDLSRTRE ETAAQVASLK KNVWLDRGTR ATFIDFSVYN AN INLFCVV RLLVEFPATG GVIPSWQFQP LKLIRYVTTF DFFLAACEII FCFFIFYYVV EEILEIRIHK LHYFRSFWNC LDV VIVVLS VVAIGINIYR TSNVEVLLQF LEDQNTFPNF EHLAYWQIQF NNIAAVTVFF VWIKLFKFIN FNRTMSQLST TMSR CAKDL FGFAIMFFII FLAYAQLAYL VFGTQVDDFS TFQECIFTQF RIILGDINFA EIEEANRVLG PIYFTTFVFF MFFIL LNMF LAIINDTYSE VKSDLAQQKA EMELSD

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II / Details: blot for 7 seconds, -1 mm offset before plunging.
DetailsSingle particles embedded in lipid nanodiscs. This sample was monodisperse.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41132 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 31000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 1500 / Average exposure time: 0.2 sec. / Average electron dose: 1.35 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 368032 / Details: semi-automated particle picking
CTF correctionSoftware - Name: CTFFIND4
Startup modelType of model: EMDB MAP
EMDB ID:

Details: TRPV1 structure
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 93805
Detailssuperresolution mode
FSC plot (resolution estimation)

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